DNA methylation is found in the genomes of diverse organisms including both prokaryotes and eukaryotes. In prokaryotes, DNA methylation occurs on both cytosine and adenine bases and encompasses part of the host restriction system. In multicellular eukaryotes, however, methylation seems to be confined to cytosine bases and is associated with a repressed chromatin state and inhibition of gene expression (reviewed, for example, in Wilson, G. G. and Murray, N. E. (1991) Annu. Rev. Genet. 25, 585-627).
In mammalian cells, DNA methylation predominantly occurs at CpG dinucleotides, which are distributed unevenly and are underrepresented in the genome. Clusters of usually unmethylated CpGs (referred to as CpG islands) are found in many promoter regions (reviewed, e.g., in Li, E. (2002) Nat. Rev. Genet. 3, 662-673). Changes in DNA methylation leading to aberrant gene silencing have been demonstrated in several human cancers (reviewed, e.g., in Robertson, K. D. and Wolffe, A. P. (2000) Nat. Rev. Genet. 1, 11-19). Hypermethylation of promoters was demonstrated to be a frequent mechanism leading to the inactivation of tumor suppressor genes (Bird, A. P. (2002) Genes Dev. 16, 6-21).
Various methods exist for experimentally determining differential methylation in individual genes (reviewed, e.g., in Rein, T. et al. (1998) Nucleic Acids Res. 26, 2255-2264). These techniques include inter alia bisulfite sequencing, methylation specific PCR (MSP), Methylight and pyro-sequencing.
One common prerequisite for performing the above techniques is the bisulfite-mediated conversion (also referred to as bisulfite modification) of the DNA to be analyzed. In particular, unmethylated cytosine residues are converted into uridine residues. The three-step reaction scheme for the bisulfite-mediated conversion from cytosine to uracil is schematically shown in FIG. 1. In brief, cytosine is sulfonated to cytosine-bisulfite under slightly acidic conditions. Hydrolytic deamination to uracil-bisulfite occurs spontaneously. The latter one is then desulfonated to uracil under basic conditions.
Since methylated cytosine residues are not converted to uridine residues, during bisulfite treatment, the DNA sequence in unmethylated CpG islands is effectively changed (C to U), while methylated DNA retains its original sequence.
However, for a valid diagnostic results based on the analysis of the DNA methylation status it is desirable that the DNA is converted with maximal efficiency, that is, ideally 100% of the unmethylated cystosine residues present in a given DNA sequence are converted to uridine residues.
Bisulfite-mediated DNA conversion is typically performed using commercially available reaction kits. In these test systems, the DNA is often incubated for a long period of time (in many cases, overnight) at a comparably high reaction temperature (e.g., 60° C.). Repeated heating steps to 95° C. are necessary during this time of incubation in order to denature the DNA. In many cases, incubation time is supposed to reach the highest DNA conversion efficiency by simply letting the reaction run for as much time as seems adequate and/or is tolerable while maintaining a certain level of DNA quality. On the other hand, it is also apparent that prolonged heating periods finally result in the degradation of DNA, and thus in a decrease in DNA yield and integrity. This may be fatal for any downstream analyses, for example, if the DNA concentration in the sample is low.
However, different sample DNAs to be analyzed or different applications likely required distinct experimental set-ups in order to achieve maximal efficiency. For example, the use of a crude lysate with unpurified DNA poses more uncertainties than a purified sample DNA. In a crude lysate, other substances are present that may potentially interact with the bisulfite salt and thus interfere with the DNA conversion reaction.
In view of the above considerations it is evident that a general approach of “one incubation time fits all” is not reasonable since it would lead to an unnecessary loss of DNA quality due to prolonged heat exposure when DNA samples are analyzed that are easy to convert (e.g., purified DNA molecules). Vice versa, the DNA in complex samples (e.g., crude lysates, body fluids, frozen biopsies) may only be converted to a rather small extent, if at all.
Currently, no methods are available that actively monitor performance and progression of bisulfite-mediated DNA conversion. However, the provision of such a method would aid to accurately determine the endpoint (i.e. 100% completion) of each individual reaction, thus enabling to switch from a generalized protocol to sample-specific reaction conditions Unnecessary and excessive heat incubation times could be avoided, thereby improving DNA quality.
Hence, there remains a continuing need for a method allowing for an accurate monitoring of the bisulfite-mediated DNA conversion overcoming the above limitations. In particular, there is a need for a corresponding method enabling the setup of individualized reaction conditions for each sample DNA analyzed, thus improving the results of differential DNA methylation analyses